For CD4+T lymphocyte activation to occur, two distinct signals must be delivered by antigen presenting cells to resting T lymphocytes (Schwartz, R. H. (1990) Science 248:1349-1356; Williams, I. R. and Unanue, E. R. (1991) J. Immunol. 147:3752-3760; Mueller, D. L. et al., (1989) J. Immunol. 142:2617-2628). The first, or primary, activation signal is mediated physiologically by the interaction of the T cell receptor/CD3 complex (TcR/CD3) with MHC class II-associated antigenic peptide and gives specificity to the immune response. The second signal, the costimulatory signal, regulates the T cell proliferative response and induction of effector functions. Costimulatory signals appear pivotal in determining the functional outcome of T cell activation since delivery of an antigen-specific signal to a T cell in the absence of a costimulatory signal results in functional inactivation of mature T cells, leading to a state of tolerance (Schwartz, R. H. (1990) Science 248:1349-1356).
Molecules present on the surface of antigen presenting cells which are involved in T cell costimulation have been identified. These T cell costimulatory molecules include murine B7-1 (mB7-1; Freeman, G. J. et al., (1991) J. Exp. Med. 174:625-631), and the more recently identified murine B7-2 (mB7-2; Freeman, G. J. et al., (1993) J. Exp. Med. 178:2185-2192). Human counterparts to the murine B7-1 and B7-2 molecules have also been described (human B7-1 (hB7-1) Freedman, A. S. et al., (1987) J. Immunol. 137:3260-3267; Freeman, G. J. et al., (1989) J. Immunol. 143:2714-2722; and human B7-2 (hB7-2); Freeman, G. J. et al., (1993) Science 262:909-911; Azuma, M. et al. (1993) Nature 366:76-79). The B7-1 and B7-2 genes are members of the immmunoglobulin gene superfamily.
B7-1 and B7-2 display a restricted pattern of cellular expression, which correlates with accessory cell potency in providing costimulation (Reiser, H. et al. (1992; Proc. Natl. Acad. Sci. USA 89:271-275; Razi-Wolf Z. et al., (1992) Proc. Natl. Acad. Sci. USA 89:4210-4214; Galvin, F. et al. (1992) J. Immunol. 149:3802-3808; Freeman, G. J. et al., (1993) J. Exp. Med. 178:2185-2192). For example, B7-1 has been observed to be expressed on activated B cells, T cells and monocytes but not on resting B cells, T cells or monocytes, and its expression can be regulated by different extracellular stimuli (Linsley, P. S. et al., (1990) Proc. Natl. Acad. Sci. USA 87:5031-5035; Linsley, P. S. et al., (1991) J. Exp. Med. 174:561-569; Reiser, H. et al. (1992); Proc. Natl. Acad. Sci. USA 89:271-275; Gimmi, C. D. et al. (1991) Proc. Natl. Acad. Sci. USA 88:6575-6579; Koulova, L. et al. (1991) J. Exp. Med. 173:759-762; Azuma, M. et al. (1993) J. Exp. Med. 177:845-850; Sansom, D. M. et al. (1993) Eur. J. Immunol. 23:295-298)
Both B7-1 and B7-2 are counter-receptors for two ligands, CD28 and CTLA4, expressed on T lymphocytes (Linsley, P. S. et al., (1990) Proc. Natl.Acad. Sci. USA 87:5031-5035; Linsley, P. S. et al., (1991) J. Exp. Med. 174:561-569). CD28 is constitutively expressed on T cells and, after ligation by a costimulatory molecule, induces IL-2 secretion and T cell proliferation (June, C. H. et al. (1990) Immunol. Today 11:211-216). CTLA4 is homologous to CD28 and appears on T cells after activation (Freeman, G. J. et al. (1992) J. Immunol. 149:3795-3801). Although CTLA4 has a significantly higher affinity for B7-1 than does CD28, its role in T cell activation remains to be determined. It has been shown that antigen presentation to T cells in the absence of the B7-1/CD28 costimulatory signal results in T cell anergy (Gimmi, C. D. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6586-6590; Boussiotis, V. A. et al. (1993) J. Exp. Med. 178:1753). The ability of T cell costimulatory molecules such as B7-1 and B7-2 to bind to CD28 and/or CTLA4 on T cells and trigger a costimulatory signal in the T cells provides a functional role for these molecules in T cell activation.
This invention pertains to novel forms of T cell costimulatory molecules. In particular, the invention pertains to isolated proteins encoded by T cell costimulatory molecule genes which contain amino acid sequences encoded by novel exons of these genes. The isolated proteins of the invention correspond to alternative forms of T cell costimulatory molecules. Preferably, these alternative forms correspond to naturally-occurring, alternatively spliced forms of T cell costimulatory molecules or are variants of alternatively spliced forms which are produced by recombinant DNA techniques. The novel forms of T cell costimulatory molecules of the invention contain an alternative structural domain (i.e., a structural domain having an amino acid sequence which differs from a known amino acid sequence) or have a structural domain deleted or added. The occurrence in nature of alternative structural forms of T cell costimulatory molecules supports additional functional roles for T cell costimulatory molecules.
The invention also provides isolated nucleic acid molecules encoding alternative forms of proteins which bind to CD28 and/or CTLA4 and isolated proteins encoded therein. Isolated nucleic acid molecules encoding polypeptides corresponding to novel structural domains of T cell costimulatory molecules, and isolated polypeptide encoded therein are also within the scope of the invention. The novel structural domains of the invention are encoded by exons of T cell costimulatory molecule genes. In one embodiment of the invention, the T cell costimulatory molecule gene encodes B7-1. In another embodiment, the T cell costimulatory molecule gene encodes B7-2.
Another aspect of the invention provides proteins which bind CD28 and/or CTLA4 and contain a novel cytoplasmic domain. T cell costimulatory molecule genes which contain exons encoding different cytoplasmic domains which are used in an alternate manner have been discovered. Alternative splicing of mRNA transcripts of a T cell costimulatory molecule gene has been found to generate native T cell costimulatory molecules with different cytoplasmic domains. The existence of alternative cytoplasmic domain forms of T cell costimulatory molecules supports a functional role for the cytoplasmic domain in transmitting an intracellular signal within a cell which expresses the costimulatory molecule on its surface. This indicates that costimulatory molecules not only trigger an intracellular signal in T cells, but may also deliver a signal to the cell which expresses the costimulatory molecule. This is the first evidence that the interaction between a costimulatory molecule on one cell and its receptor on a T cell may involve bidirectional signal transduction between the cells (rather than only unidirectional signal transduction to the T cell).
In yet another aspect of the invention, proteins that bind CD28 and/or CTLA4 and contain a novel signal peptide domain are provided. T cell costimulatory molecule genes which contain exons encoding different signal peptide domains which are used in an alternate manner have been discovered. Alternative splicing of MRNA transcripts of the gene can generate native T cell costimulatory molecules with different signal peptide domains. The existence of alternative signal peptide domain forms of T cell costimulatory molecules also suggests a functional role for the signal peptide of T cell costimulatory molecules.
An isolated nucleic acid molecule of the invention can be incorporated into a recombinant expression vector and transfected into a host cell to express a novel structural form of a T cell costimulatory molecule. The isolated nucleic acids of the invention can further be used to create transgenic and homologous recombinant non-human animals. The novel T cell costimulatory molecules provided by the invention can be used to trigger a costimulatory signal in a T lymphocyte. These molecules can further be used to raise antibodies against novel structural domains of costimulatory molecules. The novel T cell costimulatory molecules of the invention can also be used to identify agents which stimulate the expression of alternative forms of costimulatory molecules and to identify components of the signal transduction pathway induced in a cell expressing a costimulatory molecule in response to an interaction between the costimulatory molecule and its receptor on a T lymphocyte.